Skip to main content
. 2007 Jul 16;27(18):6334–6349. doi: 10.1128/MCB.00630-07

FIG. 2.

FIG. 2.

The NES in Keap1 controls nuclear export of Nrf2. (A) NIH 3T3 cells were singly transfected with an expression vector for the indicated Nrf2 or Keap1 protein. Subcellular localization of Nrf2 or Keap1 was determined by indirect immunofluorescence analysis using anti-HA for Nrf2 (panels A, D, G, J, and M) or anti-CBD for Keap1 (panels P and S). The nucleus was visualized by Hoechst staining (panels B, E, H, K, N, Q, and T). (B) Subcellular distribution of the Nrf2 or Keap1 protein in singly transfected cells (the representative image is shown in A) was determined by examining at least 100 positive cells. Percentages of cells that localized predominantly in the cytosol (C), the whole cell (W), or the nucleus (N) were presented as a bar graph. (C) NIH 3T3 cells were cotransfected with expression vectors for the indicated Nrf2 and Keap1 proteins. Subcellular localization of the Nrf2 and Keap1 proteins was determined by double-labeling indirect immunofluorescence assay using anti-HA (panels 1, 5, 9, 13, 17, 21, and 25) and anti-CBD antibodies (panels 2, 6, 10, 14, 18, 22, and 26). The nucleus was visualized by Hoechst staining (panels 3, 7, 11, 15, 19, 23, and 27). Colocalization of the Nrf2 and Keap1 proteins is indicated by the presence of yellow in the merged images (panels 4, 8, 12, 16, 20, 24, and 28). (D) Subcellular localization of the Nrf2 and Keap1 proteins in double-transfected cells (the same slides were used for C) were examined and counted in the same way as in B except that the data were collected from 100 cells that are positive for both Nrf2 and Keap1 proteins. (E) Nuclear and cytoplasmic proteins were isolated from MDA-231 cells cotransfected with expression vectors for Nrf2 and either Keap1-WT or Keap1-NES. The transfected cells were either left untreated or treated with 10 μM MG132 for 4 h prior to subcellular fractionation. Nuclear and cytoplasmic proteins derived from equal numbers of cells were electrophoresed through a 7.5% SDS-polyacrylamide gel and subjected to immunoblot analysis using anti-HA, anti-Keap1, antitubulin, or anti-lamin A antibodies. α, anti.